This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. DEVELOPMENT AND REFINEMENT OF HYBRID SPECIMEN PREPARATION METHODS FOR THE CREATION OF SPECIMEN GEOMETRIES MORE SUITABLE FOR 3-D IMAGING. The overall goal of this subproject is to develop methods to improve our ability to obtain high-resolution 3-D information from biological specimens. One of the goals during this funding period is to improve the resolution of electron tomograms of resin embedded material by overcoming tilt-induced thickness increases inherent to single-axis and double-axis tilt tomography by the creation of specimens with more suitable geometry. Our previous efforts have been directed towards devising methods for creating specimens with new shapes to work with precise total rotation tilting specimen holders being developed at the NCMIR. These methods should increase the resolution and fidelity of the tomographic reconstructions from specimens developed using methods described in aims 4A1.1 and 4A1.2. Additionally, over the last reporting period, we have been working to improve the highly promising technique of serial block face scanning electron microscopy to facilitate automated high resolution large scale 3-D imaging and reconstruction of complex tissues and organisms. As described below, we have made significant progress using serial block face scanning electron microscopy both in our technology development and collaborative programs.